The key aim was to identify novel reprogramming factors that activate the embryonic genome. We hypothesized that motifs for these transcription factors are enriched in the regulatory regions of ZGA genes. Bioinformatics motif searches identified an enrichment of SINE B1 retrotransposable elements upstream of ZGA genes. To study transcription factors such as NR5A2 that potentially bind to SINE B1, we optimized low-input genomic profiling approaches (Methods in Mol Biol 2025) and determined that NR5A2 binds to SINE B1. Using knockdown, Trim-Away and chemical inhibition, we demonstrated that NR5A2 perturbation reduced ZGA, suggesting that NR5A2 and potentially other orphan nuclear receptors are regulators of this process (Science 2022; Nat Rev Mol Cell Biol 2024). To understand NR5A2’s mechanism, we determined a cryo-EM structure of NR5A2 bound to the nucleosome, the DNA packaging unit. NR5A2 promotes partial unwrapping of nucleosomal DNA, which can contribute to generating accessible chromatin and facilitating transcription (Nat Struct Mol Biol 2024). How NR5A2 regulates distinct transcriptional programs during development remained a mystery. To address this, we profiled NR5A2 binding at multiple stages of development and found that its preferential binding to SINE B1 decreases with each embryonic cell division. NR5A2 regulates gene expression of lineage determining genes, whose products in turn co-regulate transcriptional networks during the totipotency-to-pluripotency transition (Development 2026).
Another aim was to test how chromatin-bound complexes affect genome folding. DNA is folded into loops by a process of loop extrusion that is mediated by the cohesin complex in interphase cells. Our previous work showed that cohesin is required for loops in early embryos. The progressive growth of loops is stopped when the loop extrusion machinery encounters a chromatin-bound protein called CTCF. This was the only known barrier to loop extrusion in vertebrates. Our work revealed that the replicative helicase MCM complex forms randomly placed extrusion barriers that hinder loop extrusion and affect gene expression (Nature 2022; Curr Opin Genet Dev 2024). These findings opened up the possibility to investigate the molecular properties that confer barrier function to proteins and understand more comprehensively how genome folding occurs in the complex chromatin environment.
